With the rapid increase in knowledge of the cellular mechanisms and processes that lead to disease or that are disrupted by genetic mutations, scientists can envisage potential solutions that involve the delivery of molecules into a cell that can then affect a change in a mechanism or process for therapeutic or prophylactic value. For example, several technologies to deliver nucleic acids, particularly DNA, into a cell have been described. Researchers have identified at least three requirements for accomplishing DNA delivery: 1) DNA has to be in a form that will associate with the cell membrane and be taken up by the cell; 2) the DNA has to be coupled with a molecule that is targeted to the correct types of cells, and 3) the DNA has to move into the cell compartments where it can exert a desired biological effect.
To address the first requirement, cationic lipid vectors have been developed during the last decade. Cationic vectors are a class of macromolecules that enhance the delivery of DNA by virtue of their positive charge. The cationic charge of the lipids causes an electrostatic affinity of derived liposomal preparations for the DNA and the cell membrane. Polylysine and other cationic peptides have also been developed in order to condense the DNA, but their overall efficiency relies on facilitation of the transferred DNA release from endosomal compartments (Plank, C. et al. 1994, J. Biol. Chem. 269:12918–12924; Gottschalk, S. et al. 1996, Gene Ther. 3:48–57). To address the first and third requirements, another polycationic vector, polyethyleneimine (PEI), has recently gained favor due to its intrinsic endosomolytic properties. Every third backbone atom of PEI is an amino nitrogen providing exceptionally high pH buffering capacity. In the endosome, PEI acts as an efficient “proton sponge” probably triggering osmotic swelling and disruption of endosomal vesicles which promotes efficient gene transfer demonstrated in vitro (Boussif, O., et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 7297–7301) and in vivo (Abdallah, B., et al. 1996. Hum. Gene Ther. 7:1947–1954).
However, improved targeting strategies, to address the second requirement, have been required to improve the overall efficiency of PEI among nonadherent cells (Boussif, O., et al., ibid.). Thus, each generation of cationic vectors has evolved from experimental data gained from related vectors. The efficiency of several vector classes has been improved by enhancing attachment to specific plasma membrane receptors. Those receptors are usually selected due to their naturally high abundance on the targeted cell types. The recent description of several viral vector receptors has led to strategies aimed at the manipulation of viral tropism including viral pseudotyping (Kasahara, N., et al. 1994, Science 266:1373–1376). However, the production of clinical grade virions remains arduous. Conversely, attempts have been made to artificially express viral receptors on human cells in order to increase transfer efficiencies (Bertran, J. et al. 1996, J. Virol. 70:6759–6766). Similar adaptation of lipid and polymeric vectors has been accomplished by conjugating them to ligands for known target cell receptors. While these lipid and bioconjugate vectors are relatively simple to prepare, their stability in vivo and transfection efficiency in primary cells remains low. Asialoglycoprotein receptors expressed at high levels exclusively on hepatocytes have been used in gene delivery studies (Zanta, M. A. et al. 1997, Bioconjug. Chem. 8:839–844). Folate receptors on neoplastic cells have been used for decades for the delivery of therapeutic folate analogs. Delivery of DNA to cancer cell lines via these folate receptors has been demonstrated, but transfection is limited to cells expressing the receptor at high levels (Dachs, G. U., et al. 1997, Oncol. Res. 9:313–325). The same concept of targeting highly expressed receptors has been successfully applied to the incorporation of transferrin into bioconjugates. Binding of transferrin to rapidly growing cells leads to its clustering in coated pits and eventual transfer into the cytoplasm (Schwarzenberger, P., et al. 1997, J. Virol. 71:8563–8571). In addition to natural ligands for cell surface receptors, monoclonal antibodies directed against highly expressed surface receptors have been incorporated into the design of bioconjugates (Poncet, P., et al. 1996 Gene Therapy 3:731–738). While these techniques for receptor-targeted gene delivery hold great promise, the broad application of the concept is limited by the need to develop a distinct vector for each receptor and the inevitable reliance on naturally occurring receptor molecules which are often expressed at an inadequate level on primary cells, which will be a major target for therapies. Since there is a clear correlation between the number of membrane receptors and transfection efficiency, it is an as yet elusive goal to increase the number of receptors on the cell surface as much as possible.
Thus, there is a need for a method to introduce new receptors or, alternatively to increase the number of receptors, on nucleated cells so that they can become efficient targets for delivery of therapeutic, prophylactic or diagnostic molecules. There is further a need for a universal ligand-mediated system, so that a wide range of therapeutic, prophylactic or diagnostic molecules can be delivered through the same receptor.